# Conjugative delivery of toxin genes ccdB and kil confers synergistic killing of bacterial recipients

## TL;DR

Using bacteria to deliver toxin genes via conjugation can effectively kill other bacteria, offering a new approach to combat antibiotic resistance.

## Contribution

Conjugative delivery of multiple toxin genes, rather than CRISPR-Cas9, shows stronger bacterial killing effects.

## Key findings

- Toxin gene delivery was more effective than CRISPR-Cas9 in suppressing bacterial growth.
- Combining CRISPR-Cas9 with toxin genes enhanced killing of transconjugant populations.
- Capsule production in recipient bacteria had minimal protective effect against conjugative killing.

## Abstract

The bacterial type IV secretion systems (T4SS) are medically problematic for their roles in the dissemination of mobile genetic elements or effector proteins, but they also have great potential for new antimicrobial therapies. Recent studies have deployed the T4SS subfamily of conjugation systems to deliver gene editing CRISPR/Cas systems to disrupt drug resistance genes or kill targeted bacterial recipients. However, the therapeutic potential of conjugative CRISPR/Cas delivery is compromised by mutations or host repair systems that diminish the efficiency with which CRISPR/Cas induces double-strand breaks in new transconjugants. Here, we compared the efficiencies of conjugation-based killing systems based on the delivery of CRISPR-Cas9 elements or toxin genes encoding the bacteriophage lambda Kil peptide or the F plasmid-encoded CcdB. Escherichia coli equipped with one of two efficient conjugation systems, pKM101 (IncN) or F (IncF), served as donors to mobilize plasmids carrying the cognate oriT sequence and one or more toxic elements. Overall, toxin gene delivery proved significantly more effective than CRISPR-Cas9 in killing of transconjugant population, but the highest levels of growth suppression of both E. coli and Klebsiella pneumoniae recipients were achieved by a combination of CRISPR-Cas9 plus one or two toxin genes. By contrast, capsule production conferred no or very slight protective effects on plasmid acquisition and killing of either species. We propose that the conjugative co-transfer of two or more toxic elements with distinct mechanisms of action has strong potential for growth suppression of targeted species in environmental or clinical settings.

The prevalence of antibiotic resistance emphasizes the need for alternative antimicrobial intervention strategies. We engineered Escherichia coli for conjugative transmission of plasmids encoding CRISPR-Cas9 elements or genes encoding the cell division inhibitor Kil or gyrase poisoner CcdB. Delivery of toxin genes more effectively suppressed the growth of E. coli recipients than CRISPR-Cas9, but the combinatorial delivery of CRISPR-Cas9 and a toxin gene or two toxin genes elicited the strongest killing effects. Capsule production by E. coli or Klebsiella pneumoniae recipient cells had no or little protective effect on plasmid acquisition or growth suppression. Our findings suggest that probiotic donor strains equipped for conjugative delivery of two or more toxic elements may prove effective as an alternative or adjunct to traditional antimicrobials.